Molecular Mechanisms of Class Switch Recombination

类别转换重组的分子机制

• 批准号: 8197214
• 负责人: Frederick W. Alt
• 金额: $ 42.63万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-15 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197214
• 关键词: Address; Alanine; Allergic Disease; Antibodies; Antibody Formation; Autoimmune Diseases; B lymphoid malignancy; B-Lymphocytes; Biochemical Genetics; Biological Assay; Cancer Etiology; Cells; Chromosomal translocation; Chromosome Pairing; Chromosomes; Complex; Cytidine Deaminase; Cytogenetics; DNA; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA Structure; Deamination; Disease; Double Strand Break Repair; Exons; Extrinsic asthma; Frequencies; G22P1 gene; Gene Rearrangement; Genes; Genetic; Genetic Models; Genetic Transcription; Heavy-Chain Immunoglobulins; Higher Order Chromatin Structure; IGH@ gene cluster; IgE; Immune system; Immunoglobulin Class Switching; Immunoglobulin Joining Region; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunologic Deficiency Syndromes; Immunology; Indium; Link; Lymphoma; Malignant Neoplasms; Mediating; Methods; Modification; Molecular; Molecular Conformation; Mus; Mutant Strains Mice; Mutation; Nonhomologous DNA End Joining; Oncogenes; Outcome; Pathogenesis; Pathway interactions; Phosphorylation; Process; Production; Proteins; Reagent; Regulation; Reporter; Role; Series; Serine; Single-Stranded DNA; Site; Specificity; Testing; Transgenic Organisms; Vaccines; Work; Yeasts; Zebrafish; activation-induced cytidine deaminase; chromatin immunoprecipitation; ds-DNA; endonuclease; in vitro Assay; in vivo; insight; mutant; novel; novel strategies; protein complex; repaired; replication factor A; response

This application proposes studies of the mechanisms of immunoglobulin heavy chain (IgH) class switch recombination (CSR) and Somatic Hypermutation (SHM). We have shown that Activation Induced Cytidine Deaminase (AID), the initiator of CSR, is a single strand DNA (ssDNA) specific cytidine deaminase and we employed a series of novel biochemical and genetic approaches to elucidate mechanisms by which AID gains access to transcribed double strand (ds)DNA sequences in the context transcription-generated ssDNA structures and/or certain AID modifications or co-factors. We also showed that CSR may employ general processes for synapsis of AID-initiated DNA double strand breaks (DSBs), that general DNA repair factors function in CSR, and that two distinct end-joining pathways fuse S region breaks to complete CSR. Our current proposal builds on these observations in the context of three specific Aims. Our first aim proposes use of biochemical and genetic approaches to elucidate basic mechanisms of AID function and regulation. In this regard, we developed methods to purify AID from normal B cells, in vitro assays for transcription-dependent AID deamination of dsDNA DNA, and genetic approaches to evaluate in vivo AID functions elucidated biochemically. Our second aim addresses mechanisms by which DNA sequences influence AID activity and its outcome. For these studies, we developed targeted mutation assays to replace endogenous IgH class switch (S) regions and exons encoding IgH variable regions with test sequences that will allow us to determine how substrate sequences influence activities of AID and other relevant factors in CSR and SHM. Together, the complementary biochemical and genetic assays of Aims 1 and 2 offer a powerful approach for elucidating factors and mechanisms involved in initiation and regulation of IgH CSR and SHM. A third proposed aim is to elucidate processes involved in the repair of AID induced DSBs to complete CSR. For these studies, we again have developed a large array of reagents and novel approaches, including cytogenetic methods to follow CSR related breaks in chromosomes, novel genetic approaches to study factors involved in long range synapsis of DSBs, and genetic models to elucidate DSB repair pathways that complete IgH CSR. Our proposed studies should provide novel insights into the mechanism of antibody production via IgH CSR and, therefore, be relevant to understanding immunodeficiencies, vaccine immunology, and autoimmune diseases. As CSR is required for IgE production, the work will also be relevant to understanding pathogenesis of allergic diseases and asthma. Finally, the work is relevant to B cell malignancies as they often involve chromosomal translocations that link translocated oncogenes to IgH S regions via aberrant CSR.

该应用提出了有关免疫球蛋白重链（IGH）类开关机制的研究 重组（CSR）和躯体超成熟（SHM）。我们已经表明活化诱导胞苷 CSR的引发剂脱氨酶（AID）是单链DNA（ssDNA）特异性胞苷脱氨酶，我们 采用了一系列新型的生化和遗传方法来阐明辅助的机制 在上下文转录生成的ssDNA中访问转录的双链（DS）DNA序列 结构和/或某些辅助修改或副因素。我们还表明，企业社会责任可能会采用一般 一般DNA修复因子的AID发射DNA双链断裂（DSB）的突触的过程 在CSR中的功能，并且两个不同的最终连接途径融合了S区域破裂以完成CSR。我们的 当前的提案是基于这些观察结果的三个特定目标。我们的第一个目标建议使用 生化和遗传方法阐明了援助功能和调节的基本机制。在这个 考虑到，我们开发了从正常B细胞​​中净化辅助的方法，体外测定转录依赖性 DSDNA DNA的帮助脱氨和评估体内援助功能的遗传方法阐明了 生化。我们的第二个目的是针对DNA序列影响辅助活动和 它的结果。对于这些研究，我们开发了靶向突变测定以取代内源性IGH类 开关区域和外显子编码IGH变量区域的测试序列，这将使我们能够确定 底物序列如何影响CSR和SHM中AID和其他相关因素的活动。在一起， 目标1和2的互补生化和遗传测定提供了一种强大的方法来阐明 IGH和SHM的启动和调节涉及的因素和机制。提出的第三个目标是 阐明辅助诱导DSB涉及的过程以完成CSR。对于这些研究，我们再次 已经开发了大量的试剂和新方法，包括遵循CSR的细胞遗传学方法 染色体的相关断裂，研究与涉及远程突触因素的新遗传方法 DSB和遗传模型阐明了完成IGH CSR的DSB修复途径。我们提出的研究 应该通过IGH CSR提供有关抗体生产机制的新见解，因此 与了解免疫缺陷，疫苗免疫学和自身免疫性疾病有关。就像企业社会责任一样 IgE生产所需的工作也将与了解过敏性疾病的发病机理有关 和哮喘。最后，这项工作与B细胞恶性肿瘤有关，因为它们通常涉及染色体 通过异常CSR将肿瘤基因与IGH S区域联系起来的易位。